The cranial sensory placodes are important embryonic precursors that give rise to critical structures in the vertebrate head, including olfactory epithelium, lens, acoustic and vestibular organs and the cranial sensory ganglia. In addition, migrating cranial neural crest require an interaction with placodes in order to form craniofacial cartilages properly. Despite the fact that the cranial placodes have been histologically recognized for over a century and the vital contributions to cranial sensory organs have been recognized for nearly as long, very little is known about the molecular mechanisms that specify the cranial sensory progenitor cells (SPCs) or lead to individual placode identity and differentiation. One goal of this proposal is to identify the gene regulatory network that regulates the specification of multipotent placode-derived SPCs to understand the genetic hierarchy underlying cranial sensory organ formation. Damage to placode-derived structures or congenital defects of the placodes or their common precursor tissue can have a devastating effect on an individual, profoundly impairing the sense of smell, sight, hearing, balance, taste and somatothesis of the face. Recent studies have connected mutations in genes that play a central role in placode development to several craniofacial disorders. A second goal of this proposal is to identify new candidate genes that underlie craniofacial birth defects that involve placode-derived sensory structures. To accomplish these goals, we shall focus our studies on the regulation of the transcription factor Six1 for two reasons. First, mutations of Six1 result in the branchio-otic syndrome 3 (BOS3, OMIM 608389), which is characterized by craniofacial defects and hearing loss. Second, Six1 knock-out in mouse and Six1 knock-down in Xenopus and chick result in severe defects in several placode-derived structures. To accomplish our two main goals, we shall determine: 1) what genes directly regulate Six1 expression using bioinformatics, in vivo and biochemical assays; 2) the epistatic relationships between Six1 and its downstream targets, relying on microarray data obtained from two different animal models; and 3) which of the genes that act down-stream of Six1 are direct targets. The proposed experiments represent a close collaboration between the Moody and Streit labs; our labs have worked in parallel on similar topics in placode development using two different animal models (Xenopus and chick, respectively). We now propose to combine our expertise to create a gene regulatory network that regulates cranial sensory precursor specification and placode development. Using two different, powerful animal models will provide the strongest information for conserved gene regulation across vertebrates that will have the most relevance to human craniofacial syndromes. This research project utilizes classical embryological approaches in the hands of two experts renowned for their studies of placode development and combines them with cutting edge technological advances. This combination of experimental strengths predict that important information regarding normal development as well as discovery of novel genetic causes of craniofacial birth defects will be forth coming from this project.